Transforming growth factor b (TGFb) is prototypic of a family of cytokines with immunoregulatory properties that are critical to the maintenance of normal immunological homeostasis. Targeted disruption of the TGFb1 gene in mice results in a phenotype characterized as a progressive multifocal inflammatory process typical of autoimmune disease. The inflammatory response in the TGFb-knockout is lymphocyte mediated demonstrating the vital role of TGFb in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance. This role of TGFb in the maintenance of self tolerance through clonal deletion of B lymphocytes by programmed cell death or apoptosis is fairly well established but poorly characterized. In this proposal we wish to determine the molecular mechanisms by which TGFb aids in maintenance of self tolerance through its induction of apoptosis in B lymphocytes. We have obtained preliminary results demonstrating that TGFb-induced apoptosis in B-lymphocytes is in part mediated through its induction of the pro-apoptotic form of the Bcl-2 family member Bim, Bcl-2 interacting mediator of cell death. This is the first demonstration that direct addition of a pro-apoptotic inducer (TGFb), and not withdrawal of a survival factor results in increased Bim expression. TGFb induction of Bim is transcriptionally mediated through Smad3 and is abrogated by activation of the survival pathway induced through the CD40 receptor. Since it has previously been demonstrated that Bim expression is regulated by the forkhead family (FKHD) of transcription factors (specifically FKHR-L1) and that CD40 couples to activation of the PI3-K/Akt pathway which regulates FKHR-L1 transcriptional activity, we hypothesize that TGFb induced Bim expression and apoptosis in B lymphocytes is mediated through a cooperativity between the FKHR-L1 and the Smad3 signaling pathway. We also wish to isolate novel and previously uncharacterized mediators of TGFb-induced apoptosis using a gene inactivation/mutagenesis approach. [unreadable] [unreadable]